I am facing difficulties in getting proper contrast images of doxorubicin based bilayer liposomes using Cryo Transmission Electron microscopy (120C Talos thermofisher). If anyone who is using Cryo TEM for liposomes please answer following queries
1. What concentration will be better for getting better liposomes images. I am preparing 1:10 and 1:20 ratios for liposomes suspension and dextran buffer.
2. How to prevent ice formation on TEM grid during vitrification process using vitrobot.
3. During acquiring images how to focus properly to get better contrast of the micrographs.
4. Which TEM grid is better for Cryo TEM of liposomes.
5. What are the additional precautions we can take during liposomes imaging using Cryo TEM.
I will highly appreciate your answers on the above queries. Thank you in advance.
Question 1. 1:100 may be better as those concentrations you are preparing may be too concentrated.
Question 3. To get better images try starting with lower magnification and then increase gradually.
Ice formation can be prevented by absorbing excess liquid using a filter paper.
Thank you Chinazom Agbo for your response. I am trying with lower magnification but the contrast of the images are poor. The micrographs doesn't looks clear. How can we improve contrast of these micrographs.
I am using blotting paper for removing excess liquid during vitrification process using vitrobot but unfortunately still ice formation is there.
I have tried preparing liposomes samples using negative staining method with urenyl sulphate 2% solution. But the liposomes starts burning due to electron bombardment. We tried low dose method also but not much success.. still trying..